Methods and devices for digital pcr

ABSTRACT

A method comprises flowing a plurality of sample droplets in a continuous flow of a carrier fluid, immiscible with the sample droplets, such that the droplets are separated from each other by the carrier fluid, wherein an average number of copies of target nucleic acid contained in each droplet the plurality of sample droplets is one or fewer. The method may further comprise subjecting the droplets to thermal cycling sufficient to allow amplification of the target nucleic acid, and detecting one or more of the presence or absence of amplified target nucleic acid in the droplets.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 16/812,987, filed Mar. 9, 2020, which iscontinuation application of U.S. patent application Ser. No. 15/488,819,filed Apr. 17, 2017 (now U.S. Pat. No. 10,626,451), which is adivisional application of U.S. patent application Ser. No. 12/539,343,filed Aug. 11, 2009 (now U.S. Pat. No. 9,631,230), which claims priorityto U.S. Provisional Application No. 61/088,142, filed Aug. 12, 2008,each of which are incorporated by reference in their entirety herein.

TECHNICAL FIELD

The invention relates to methods and devices for conducting nucleic acidamplification reactions, including the polymerase chain reaction (PCR).

BACKGROUND

PCR is a molecular amplification method routinely practiced in medicaland bioresearch settings for a variety of tasks, such as the detectionof hereditary diseases, the identification of genetic fingerprints, thediagnosis of infectious diseases, the cloning of genes, paternitytesting, and other types of nucleic acid analysis. For a review of thePCR methodology, see, e.g., PCR Protocols (Methods in Molecular Biology)by Barlett and Stirling (eds.), Humana Press (2003); and PCR byMcPherson and Moller, Taylor & Francis (2006).

Digital PCR is a technique that allows amplification of a single DNAtemplate from a minimally diluted sample, thus, generating ampliconsthat are exclusively derived from one template and can be detected withdifferent fluorophores or sequencing to discriminate different alleles(e.g., wild type vs. mutant or paternal vs. maternal alleles). For areview of the digital PCR methodology, see, e.g., Pohl et al., ExpertRev. Mol. Diagn., 4(1):41-7 (2004). The basic premise of the techniqueis to divide a large sample into a number of smaller subvolumes(segmented volumes), whereby the subvolumes contain on average a singlecopy of a target. Then, by counting the number of positives in thesubvolumes, one may deduce the starting copy number of the target in thestarting volume. Most commonly, multiple serial dilutions of a startingsample are used to arrive at the proper concentration in the subvolumes,the volumes of which are typically determined by a given PCR apparatus.This additional step increases the number of samples to be processed. Aset of subvolumes may be tested that statistically represents the entiresample to reduce that number. However, under certain conditions, it maybe necessary to detect very lowly expressed genes, resulting in a largenumber of blank segmented volumes and, thus, a large number ofsubvolumes to be evaluated. While making a sample more concentrated is apossibility in this case, doing so may introduce significant variabilityand losses (see, e.g., N. Blow, Nature Methods, 4:869-875 (2007). Inaddition, a more concentrated sample means that more sample is necessaryto begin with.

Further considerations suggest that decreasing the volume of theamplification reaction might improve sensitivity for detecting a singlemolecule. For example, the TaqMan® assay requires near-saturatingamounts of PCR amplification product to detect fluorescence. PCRreactions normally saturate at about 1011 product molecules/microliterdue, in part, to reannealing of product strands. To reach thisconcentration of product after 30 cycles in a 10 μl PCR requires atleast 103 starting template molecules. If the volume of the PCR werereduced to ˜10 nanoliters, then a single molecule could generate therequired product to be detected by the TaqMan® assay. Attempts have beenmade to miniaturize PCR volumes (for a review, see, e.g., Zhang et al.,Nucl. Acids Res., 35(13):4223-4237 (2007)). Nevertheless, as samplevolumes decrease, amplification becomes increasingly more prone tobiochemical surface absorption problems due to the increasingsurface-to-volume ratio, as well as potential other sources ofvariability.

Therefore, there exists a need for methods and devices for accuratelydetecting or quantifying target copy numbers, including by means of thedigital PCR.

SUMMARY OF THE INVENTION

The invention provides methods of conducting a nucleic acid reaction,including methods for performing digital PCR using “droplet-in-oil”technology, wherein a sample is segmented into droplets placed to acontinuous flow of carrier fluid through a microfluidic channel. Oneexample of such technology is described in PCT Pat. Appln. Pubs. WO2007/091228 (corresponding U.S. Ser. No. 12/092,261), WO 2007/091230(U.S. Ser. No. 12/093,132); and WO 2008/038259, and in the Examples. Insome of these systems, termed “continuous flow PCR,” the droplets arefully wrapped in the carrier fluid throughout the reaction anddetection. The invention is based, at least in part, on the realizationthat sample droplets of 10-500 nl provide advantages for a PCR analysisof lowly expressed targets. Various aspects of the invention aredescribed below.

In certain embodiments, the methods include:

-   -   a) providing a starting sample comprising a target nucleic acid        to be detected;    -   b) segmenting at least part of the sample to provide a set of        sample droplets in a continuous flow of immiscible carrier fluid        (e.g., oil) through a channel (e.g., a capillary), each of said        droplets containing on average about one or fewer copies of a        target nucleic acid and reagents sufficient to perform a        polymerase chain reaction;    -   c) passing the droplets through a plurality of thermal zones        thereby allowing the target nucleic acid, if present, to be        amplified in each droplet; and    -   d) detecting the presence or absence of, and/or determining the        amount of, the amplified target nucleic acid in the droplets        while in the flow.

The sample droplets have volumes of 0.1 pl-500 nl, preferably, 10-500nl, more preferably, 30-350 nl, while the starting sample volumes are0.05-5000 μl, preferably, 5-3500 μl. These volumes may include volumesof reagents (e.g., primer solution) added prior to the detection step.In certain embodiments, the droplets are spherical. In some embodiments,the droplets created by segmenting the starting sample are merged with asecond set of droplets comprising one or more primers for the targetnucleic acid, thereby producing the final droplets for the amplificationreaction.

In some embodiments (“real-time detection”), the step of detecting ordetermining the amount is performed at multiple thermal cycles, therebymonitoring the amount of amplified target nucleic acid throughout thecycles, for example, in performing qPCR or real time PCR. Typically, thethermocycling is performed for at least a number of cycles required toreach the near-saturation level of the amplification. The number ofcycles depends on the concentration of the target and other conditionsand is typically between 20 and 40.

In preferred embodiments (“end-point detection”), the step of detectingor determining the amount is performed after the near-saturation pointis reached. For the end-point detection, the starting copy number of thetarget nucleic acid may be determined by counting the number ofpositively amplified droplets in a given set of droplets.

The number of droplets in a set being analyzed is such that theircombined volume is representative of the starting sample. The set ofdroplets contain the entire starting sample or only its part, dependingon the number of copies of the target nucleic acid present or suspectedto be present in the starting sample. The starting concentration of atarget nucleic acid may be adjusted by diluting or by concentrating thestarting sample. In illustrative embodiments, the set of dropletscontains a train of 10 droplets, and 0.005 ng/μl cDNA in the startingsample.

The invention further provides methods of processing a plurality ofstarting samples in parallel, wherein at least some of the startingsamples have a) a varying concentration of the target nucleic acidand/or b) varying target nucleic acids. In some embodiments, sets ofdroplets from different starting samples form a train of alternatingdroplets in the continuous flow of the carrier fluid in the channel.These and other embodiments of the invention are described in detailbelow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates amplification curves for a serially diluted sample atfour concentrations of GAPDH cDNA: 5, 0.5, 0.05 and 0.005 ng/μl. Theamplification was performed as described in Example 1. The intersectionof each amplification curve with the threshold, as set at 0.3 here,defines the Ct value.

FIG. 2 shows Ct value as a function of concentrations based on theresults shown in FIG. 1 . The slope of the line allows the amplificationefficiency to be measured.

FIGS. 3A-3B show fluorescence signals from four sets of ten dropletswith decreasing template concentrations as shown in FIG. 1 . The plotscompare fluorescence measurements taken at cycle 7 (FIG. 3A) and 42(FIG. 3B).

FIGS. 4A-4C show amplification traces based on the study described inExample 2. FIG. 4A shows droplet fluorescence traces of No TemplateControl (NTC) droplets followed by seven increasing sampleconcentrations at cycle 42. FIG. 4B shows an amplification curve of ten1650 pg/μl droplets. FIG. 4C illustrates the standard curve generatedfrom Ct data showing near single molecule detection in 300 nl droplets.

FIG. 5 demonstrates a typical PCR thermal cycling trace for a set of3,000 droplets from a 300 μl sample having 0.005 ng/μl cDNA, performedas described in Example 3.

DETAILED DESCRIPTION General Methods

The invention provides methods of conducting a nucleic acidamplification reaction, such as PCR in a sample containing or suspectedto contain a target nucleic acid to be detected. Although the methodsdescribed here employ the PCR as an amplification method of choice,alternative techniques of nucleic acid amplification may similarly beused in place of the PCR. Such techniques include for example, theligase chain reaction (LCR), the transcription based amplificationsystem (TAS), the nucleic acid sequence-based amplification (NASBA), thestrand displacement amplification (SDA), rolling circle amplification(RCA), hyper-branched RCA (HRCA), etc.

In general, the invention relates to the so-called “digital PCR” andsimilar methods that allow one to quantify the starting copy number of anucleic acid template, by segmenting the starting sample to smallerreaction volumes, most of which contain one copy of the target or fewer.

Methods of the invention may be used for determining the presence of theamount of a nucleic acid target, and for example, in gene expressionanalysis and is especially useful for lowly expressed genes.

Generally, the methods of the invention include at least the followingsteps:

-   -   a) providing a starting sample comprising a target nucleic acid        to be detected;    -   b) segmenting at least part of the sample to provide a set of        sample droplets in a continuous flow of immiscible carrier fluid        (e.g., oil) through a channel (e.g., a capillary), each of said        droplets containing on average about one copy or fewer copies of        a target nucleic acid and reagents sufficient to perform a        polymerase chain reaction;    -   c) passing the droplets through a plurality of thermal zones        thereby allowing the target nucleic acid, if present, to be        amplified in each droplet; and    -   d) detecting the presence or absence of, and/or determining the        amount of, the amplified target nucleic acid in the droplets        while in the flow.

In some embodiments (“real-time detection”), the step of the detectingor determining the amount is performed at multiple thermal cycles,thereby monitoring the amount of amplified target nucleic acidthroughout the cycles, for example, in performing qPCR or real time PCR.Typically, the thermocycling is performed for at least a number ofcycles required to reach the near-saturation level of the amplification.The number of cycles depends on the concentration of the target andother conditions and is typically between 20 and 40. The dependence ofCt on the target concentration is illustrated in Example 1. In some suchembodiments, a Ct value for the target nucleic acid is determined bydetecting the course of amplification at each cycle. The “real-time”detection may be used for constructing the standard curve as well as forquantifying targets in test samples.

In preferred embodiments (“end-point detection”), the step of thedetecting or determining the amount is performed after thenear-saturation point is reached. For the end-point detection, thestarting copy number of the target nucleic acid may be determined bycounting the number of positively amplified droplets in a given set ofdroplets.

Droplets and Samples

The starting sample contains (or is suspected to contain) at least onetarget nucleic acid. As used herein, the term “starting sample” refersto the sample from which droplets are generated. For example, thestarting sample may be placed in a well in a conventional 384-wellplace, from which sample droplets are drawn. The starting sample volumesmay vary, and may be, for example, 0.05-5000 μl, preferably, 5-3500 μl,e.g., 5-1000 μl, 50-500 μl, 100-350 μl. In some embodiments, dropletscreated by segmenting the starting sample are merged with a second setof droplets comprising one more primers for the target nucleic acid toproduce final droplets. In some embodiments, the starting samplecontains at least 2, 5, 10, 100, 500, 1000 or more copies of the targetnucleic acid.

The sample droplets have volumes of 0.1 pl-500 nl, preferably, 1 pl-500nl, 10 pl-500 nl, 100 pl-500 nl, 1-500 nl, or 10-500 nl, morepreferably, 30-350 nl. In certain embodiments, sample droplets havevolumes of 50-500, 100-500, 150-500, 200-500, 50-400, 100-400, 150-400,200-400, 50-300, 100-300, 150-300, 200-300, or 150-250 nl. These volumesmay include volumes of reagents (e.g., primer solution) added prior tothe detection step. In some embodiments, the droplets are spherical,while in other embodiments, the droplets are elongated along the axis ofthe channel.

The number of droplets in a set being analyzed is such that theircombined volume is representative of the starting sample. The set ofdroplets contain the entire starting sample or only its part, dependingon the number of copies of the target nucleic acid present, or suspectedto be present, in the starting sample. For example, a set of dropletscontaining, in total, 10% of the starting sample volume may beconsidered representative for a starting sample containing 100 copies ofthe target nucleic acid. Therefore, depending on the expected number ofthe set of analyzed droplets, the sets may contain several droplets toseveral thousand droplets. In some embodiments, the set of droplets fora given target nucleic acid contains, e.g., 5-10000 droplets, e.g.,100-5000, 5-1000, 100-500, 5-50, 6-30, 10-25, 8 or more, or 10 or moredroplets. In other embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% of the starting sample, or the entire starting sample,is segmented into droplets.

The standard digital PCR aims at determining the dilution of the sampleat which only half of segmented volumes are positive. This dilutionindicates that the target nucleic acid is diluted on average to ½ persegmented volume. The present method allows one to analyze a muchgreater number of segmented volumes, many of which may be blank. In someembodiments, at least 30%, 40%, 50% 60%, 70%, 80%, 90%, 95%, 99% or moredroplets in a set do not contain a target nucleic acid. For instance,for end-point detection, fewer than 50% (e.g., 30%, 20%, 10%, 5% orless) of droplets in the set of 10 or more (e.g., 20, 50, 100, 1000,5000, or 10000) are positively amplified. Accordingly, in someembodiments, regardless of volume, the starting sample contains onegenome equivalent of nucleic acid or less. In certain applications, itmay be assumed that the mass of DNA is ˜3 pg per genome (e.g., 3.3pg/genome). F

A sample may be divided into replicates (e.g., duplicates, triplicates,etc.), in which the expression levels are measured. The sample may bederived from the same source and split into replicates prior toamplification. Additionally, one may create serial dilutions of thesample. Replicate and dilution samples may be analyzed in a serial or aparallel manner. In a parallel processing system, sets of dropletscorresponding to separate starting samples form a sequence ofalternating droplets which pass through a thermal cycler, where dropletsare being amplified, for example, as described in WO 2008/038259. Aplurality of starting samples with varying concentrations of the targetnucleic acid and/or varying target nucleic acids may be processed inthis manner in parallel.

A sample may contain material from obtained cells or tissues, e.g., acell or tissue lysate or extract. Extracts may contain material enrichedin sub-cellular elements such as that from the Golgi complex,mitochondria, lysosomes, the endoplasmic reticulum, cell membrane, andcytoskeleton, etc. In some embodiments, the biological sample containsmaterials obtained from a single cell. Biological samples may come froma variety of sources. For example, biological samples may be obtainedfrom whole organisms, organs, tissues, or cells from different stages ofdevelopment, differentiation, or disease state, and from differentspecies (human and non-human, including bacteria and virus). The samplesmay represent different treatment conditions (e.g., test compounds froma chemical library), tissue or cell types, or source (e.g., blood,urine, cerebrospinal fluid, seminal fluid, saliva, sputum, stool), etc.Various methods for extraction of nucleic acids from biological samplesare known (see, e.g., Nucleic Acids Isolation Methods, Bowein (ed.),American Scientific Publishers (2002). Typically, genomic DNA isobtained from nuclear extracts that are subjected to mechanical shearingto generate random long fragments. For example, genomic DNA may beextracted from tissue or cells using a Qiagen DNeasy Blood & Tissue Kitfollowing the manufacturer's protocols.

In case of the RNA analysis (e.g., mRNA, siRNA, etc.), for example, asin the case of gene expression analysis, the nucleic acid is initiallyreverse-transcribed into cDNA prior to conducing the PCR. This type ofPCR is commonly referred to as “RT-PCR” and is illustrated in theExamples.

Droplet-in-Oil Systems

Any suitable device may be used to practice the methods of theinvention. Generally, a PCR device contains a sample preparation system,a thermocycler, and a detection unit. During sample preparation, thesample is segmented into droplets which are wrapped in immiscible fluid(e.g., silicone oil, mineral oil) which continuously flows through thechannel, such as a capillary having a circular cross-section. The oil,enveloping each droplet, avoids cross contamination between thesequential droplets and carry-over contamination. The sample may bepre-mixed with the primer, or the primer may be added to the droplet. Insome embodiments, droplets created by segmenting the starting sample aremerged with a second set of droplets comprising one or more primers forthe target nucleic acid in order to produce final droplets. The mergingof droplets can be accomplished using, for example, one or more liquidbridges as described in WO 2007/091228 (U.S. Ser. No. 12/092,261), WO2007/091230 (U.S. Ser. No. 12/093,132) and WO 2008/038259, cited above.

A queue of droplets from the preparation system may be passed throughthe thermal cycler. The velocity of the sample through the device isdefined by the control of the velocity of the carrier fluid iscontrolled by an external pumping system. The sample undergoes the samethermal cycling and chemical reaction as it passes through Namplification cycles of the complete thermal device. This results in amaximum two-fold amplification after each cycle and a totalamplification of I(1+E)^(N) where I is the initial product, E is theefficiency of the reaction and N is the number of cycles. Fluorescentprobes are contained in each sample droplet. The fluorescence level isdetected in each droplet at each cycle, e.g., in the case of real-timePCR. This may involve the use of fluorescent probes, such as Taqman®probes, and intercollating fluorescent dyes, such as SYBR Green andLCGreen®, as described in, e.g., in U.S. Pat. Nos. 5,723,591 and5,928,907; www.idahotech.com; Gudnason et al., Nucleic Acids Res.,35(19):e127 (2007); and in the Examples.

An exemplary system for use with the method of the invention isdescribed, for example, PCT Patent Application Pubs. WO 2007/091228(U.S. Ser. No. 12/092,261); WO 2007/091230 (U.S. Ser. No. 12/093,132);and WO 2008/038259. One such system is made by Stokes Bio(www.stokebio.ie). Other exemplary systems suitable for use with themethods of the invention are described, for example, in Zhang et al.Nucleic Acids Res., 35(13):4223-4237 (2007) and include those made byFluidigm (www.fluidigm.com), RainDance Technologies(www.raindancetechnologies.com), Microfluidic Systems(www.microfluidicsystems.com); Nanostream (www.nanostream.com); andCaliper Life Sciences (www.caliperls.com). For additional systems, see,e.g., Wang et al., J. Micromech. Microeng., 15:1369-1377 (2005); Jia etal., 38:2143-2149 (2005); Kim et al., Biochem. Eng. J., 29:91-97; Chenet al., Anal. Chem., 77:658-666; Chen et al., Analyst, 130:931-940(2005); Munchow et al., Expert Rev. Mol. Diagn., 5:613-620 (2005); andCharbert et al., Anal. Chem., 78:7722-7728 (2006); and Dorfman et al.,Anal. Chem, 77:3700-3704 (2005).

The following Examples provide illustrative embodiments of the inventionand do not in any way limit the invention.

EXAMPLES Example 1

Measurement of qPCR Amplification Efficiency by Serial Dilution

Total RNA is extracted from cultured cells, reverse transcribed intocDNA and used as the template for the qPCR reaction. The startingconcentration of the template is 5 ng/μl which is then diluted 10-foldto 0.5 ng/μl. This 10-fold dilution is repeated yielding samples withfour concentrations of cDNA template: 5 ng/μl, 0.5 ng/μl, 0.05 ng/μl,and 0.005 ng/μl. The resulting amplification curves, obtained using aStokes Bio device (www.stokesbio.ie), are shown in FIG. 1 . As seen fromthe figure, for lower starting template concentrations, more cycles ofPCR are necessary to bring the fluorescence signal to the thresholdlevel. In fact, if the PCR reaction was 100% efficient a 0.5 ng/μlsample would require 3.32 more cycles of PCR to reach the same thresholdas a 5 ng/μl sample (10 times the 0.5 ng/μl sample). The fractionalcycle at which the amplification curve reaches the threshold is calledthe Ct value: a measure of the gene expression of the sample. Thethreshold is set at 0.3 read off of the corresponding Ct values for eachset of amplification curves in FIG. 1 . The Ct value is plotted againstthe cDNA concentration in FIG. 2 . Analysis of this data implies thatthe slope of the best fit line is −3.53±0.13, estimating theamplification efficiency to be 92%±4%, which is within the rangeexpected of qPCR on a standard PCT system, such as Applied Biosystems'7900HT.

FIG. 3A shows the fluorescence signal from cycle 7 of a 50 cycleamplification using the Stokes Bio device. Each of the small spikesrepresents a droplet passing. The far left spike represents the leaddroplet. The concentrations of each set of droplets range from 0.005 to5 ng/μl, as indicated in the figure. Two aspects of the data are clearlyshown—there is background fluorescence from the oil filled capillary,and background signal from each droplet—both are constant. FIG. 3B showsthe same droplets at cycle 42 when amplification is complete. Note thatthe signal from each droplet in a set of ten varies fromdroplet-to-droplet. This is because the optical system for each channelis not identical. This effect is easily normalized out during dataprocessing. At the highest level of dilution (0.005 ng/μL), only fourout of ten droplets have amplified. This is a statistical effect,expected for this level of template dilution and droplet volume. Thedata processing algorithm is designed to take this into account.

Example 2

High Throughput qPCR Performance Validation

The analysis of gene expression is an essential element of functionalgenomics, and qPCR-based expression profiling is the gold standard forthe precise monitoring of selected genes. Gene expression relies uponthe reverse transcription of mRNA to cDNA. It is, however, generally notpossible to use cDNA as a standard for absolute quantification of mRNAbecause there is no control for the efficiency of the reversetranscription step. This Example presents Stokes Bio's amplificationrepresentative performance data from genomic DNA (gDNA), a commonly usedin standard qPCR.

A TaqMan® RNase P gene primer and probe set is used to evaluateinstrument performance. The RNase P gene is a single-copy gene encodingthe RNA moiety for the RNase P enzyme. Several two-fold dilutions arecreated from a stock of a known gDNA copy number. These dilutions wereused to prepare complete qPCR reactions for amplification in the StokesBio instrument. Table 1 shows gDNA template concentrations withcorresponding mean Cts and estimated starting copy numbers for each ofthe seven reaction sets. A no template control (NTC) is also includedand showed no amplification. FIG. 4A shows droplet fluorescence tracesof three NTC reactions with seven sets of ten 300 nl droplets. Each10×replicate set was taken from a different concentration sample. Theinset plot shows excellent data resolution for each 300 nl droplet. FIG.4B shows a normalized amplification plot of the most concentratedreaction set with a Ct of 26.4. FIG. 4B shows a standard curve takenfrom the Ct data in Table 1. Error bars indicate the Ct range in each ofthe 10×replicate sets. Ct variability is attributable to Poisson noiseor a variation in the number of starting copies from one droplet toanother at a given concentration.

TABLE 1 Copies* gDNA in Standard per 300 nl pg/μl Mean Ct DeviationReaction 1650 26.4 0.11 150 825 27.7 0.16 75 412 28.9 0.35 37.5 206 29.80.44 18.75 103 30.9 3.4 9.4 51.5 31.6 0.76 4.7 25.75 32.47 0.76 2.34*Determined using 1 copy per 3.3 pg

Example 3 Digital PCR End-Point Detection

The premise for this technique is to divide a large volume into adiscreet number of smaller volumes reducing the number of copies in eachsample, following the amplification process to perform fluorescencedetection on the emerging droplets. The resulting total number ofdroplets with amplification can then be used to determine starting copynumber. It also can be used for rare target detection wherein thestatistic probability of amplification is increased for the rare targetas the number of background molecules is reduced by the division.

Using a statistical prediction model the probability of the distributedtarget molecules in the segment droplets can be generated. This isparticularly of benefit for low concentration samples as it provides aprediction of the number of droplets containing molecules and thus thenumber of droplets expected to fluoresce.

The binomial distribution model employed is a discrete probabilitydistribution that arises in many common situations. The recognizedexample of binomial distribution is counting the number of heads in afixed number of independent coin tosses, while in this case, it iscounting the number of copies in a fixed known number of dropletscreated from the original sample. In a series of n independent trials,or n independent copies, each trial or copy results in a success (theoutcome that is counted) or failure. In other words, each copy has twopossible outcomes, it enters the monitored droplet or not. For a samplesegmented into 3000 droplets, each copy has the same probability, p, ofsuccess, 1 in 3000, for each monitored droplet. The binomialdistribution model counts the number of successes in a fixed number oftrials. Binomial distribution is completely determined by twoparameters; n, the number of cDNA copies in the main volume, and p, thesuccess probability common to each copy. As a consequence, knowing thenumber of copies and the number of droplets allows a binomialdistribution to be used.

FIG. 5 demonstrates a typical trace for a 0.005 ng/μl sample. The datademonstrated here uses a 300 μl sample volume which is divided into˜3000 droplets of 100 nl droplet volumes.

In another experiment, digital PCR involves amplifying a single DNAtemplate from minimally diluted samples, generating amplicons that areexclusively derived from one template. It transforms an exponential,analog signal obtained from conventional PCR to linear, digital signals,thus allowing statistical analysis of the PCR product.

To determine the mass of genomic DNA which corresponds to copy numbersof target nucleic acid sequences, the following formula was used:

N=(m×N _(A))/M

M=n×1.096×10⁻²¹ g/bp

where n is the number of base pairs, m is the mass of DNA, N_(A) isAvogadro's number (6.02×10₂₃ bp/mol) and M is the average molecularweight of a base pair.

A series of 2-fold dilutions of human genomic DNA 10 ng/μl was carriedout. The gene of interest was the RNAse P gene which exists as a singlecopy per haploid genome. Based on the above formulae, the copy numbersof the RNAse P gene per 350 nl droplet are shown in Table 2.

TABLE 2 RNAse P Copy gDNA Rxn Cone. Copies Copies/ No Mass (pg) Vol.pg/μL 20 μl 350 nl 10000 33000 5 6600 10000 175.00 5000 16500 5 33005000 87.50 2500 8250 5 1650 2500 43.75 1250 4125 5 825 1250 21.88 6252062.5 5 413 625 10.94 312.5 1031.25 5 206 312.5 5.47 156.25 515.625 5103 156.25 2.73 78.125 257.8125 5 52 78.125 1.37 39.0625 128.90625 5 2639.0625 0.68

Mass DNA/genome was assumed to be 3.3 pg/genome. PCR amplification wascarried out on the Stokes HTI on a series of 10 No Template Controls(NTCs) followed by 10 droplets of 350 nl volume at each copy number.Results indicate that at low copy numbers, discrete amplification ofdroplets occurred. S-curve analysis of this amplification seemed toindicate that threshold cycle (Ct) values clustered around distinct Ctvalues

All publications, patents, patent applications, and biological sequencescited in this disclosure are incorporated by reference in theirentirety.

1. (canceled)
 2. A method of quantifying a target nucleic acid of asample using a system comprising a sample preparation component, athermal cycler component, and a detection component, the methodcomprising: in the sample preparation component: drawing sample fromindividual wells of a sample holder, the sample comprising nucleic acidand reagent for amplification of target nucleic acid; forming a set ofdroplets by flowing an immiscible carrier fluid into contact with a flowof the sample drawn from the wells of a sample holder and therebysegmenting the sample into the set of droplets; subjecting the set ofdroplets to an amplification reaction by thermal cycling the set ofdroplets in the thermal cycler component; after the subjecting, flowingindividual droplets of the set of droplets to a detection region of thedetection component; detecting as each individual droplet flows past thedetection region for a presence or an absence of an amplificationproduct of the target nucleic acid; calculating a total number of theindividual droplets detected to contain the amplification product; anddetermining the starting number of copies of the target nucleic acidbased on the calculated total number.
 3. The method of claim 2, whereinthe amplification reaction comprises one or more of a polymerase chainreaction (PCR), a ligase chain reaction (LCR), a transcription basedamplification system (TAS), a nucleic acid sequence-based amplification(NASBA), a strand displacement amplification (SDA), a rolling circleamplification (RCA), and a hyper-branched RCA (HRCA).
 4. The method ofclaim 2, wherein the amplification reaction is performed until anear-saturation concentration of the amplification product is producedin each droplet containing the amplification product.
 5. The method ofclaim 2, wherein the amplification reaction comprises thermal cyclingthe set of droplets through multiple thermal cycles.
 6. The method ofclaim 5, wherein the multiple thermal cycles ranges from 20 to 40thermal cycles.
 7. The method of claim 5, wherein the detecting isperformed at one or more of the thermal cycles.
 8. The method of claim5, wherein the detecting occurs at each of the multiple thermal cycles.9. The method of claim 2, further comprising altering a concentration ofthe sample by a dilution factor, wherein determining the starting numberof copies of the target nucleic acid comprises use of the dilutionfactor.
 10. The method of claim 2, wherein at least some droplets of theset of droplets do not contain any copy of the target nucleic acid. 11.The method of claim 10, wherein from 50% to 90% of droplets in the setof droplets do not contain any copy of the target nucleic acid.
 12. Themethod of claim 2, wherein the target nucleic acid comprises DNA. 13.The method of claim 12, wherein the DNA comprises genomic DNA.
 14. Themethod of claim 2, wherein the sample comprises cDNA.
 15. The method ofclaim 14, further comprising performing reverse transcription of mRNA ofthe sample to produce the cDNA.
 16. The method of claim 2, wherein theset of droplets comprises a reagent for the amplification reaction. 17.The method of claim 16, wherein the reagent comprises one or moreprimers.
 18. The method of claim 2, wherein the sample contains at least10 copies of the target nucleic acid and at least 10% of the sample issegmented.
 19. The method of claim 2, wherein a volume of each dropletof the set of droplets ranges from 0.1 μl to 500 nl.
 20. The method ofclaim 2, wherein the set of droplets comprises at least 5 droplets. 21.The method of claim 2, wherein the detecting comprises detecting theamplification product in fewer than 50% of droplets in the set ofdroplets.
 22. The method of claim 2, wherein each droplet of the set ofdroplets are wrapped in and carried by a continuous flow of theimmiscible carrier fluid during the amplification reaction anddetecting.